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PubMed2002

Identification of critical molecular interactions mediating heparin activation of antithrombin: implications for the design of improved heparin anticoagulants.

The serpin, antithrombin, and its polysaccharide activator, heparin, are essential anticoagulant regulators of blood-clotting cascade proteases and thereby critical for maintaining hemostasis. The relative importance of the molecular interactions that mediate heparin binding to and activation of antithrombin, and the dynamics of how they are established, have recently been revealed from the effects of mutagenesis of heparin-binding residues of antithrombin and of modifications of the specific pentasaccharide-binding region in heparin. One residue, Lys 125, is pivotal for antithrombin to recognize and bind the nonreducing-end trisaccharide of the pentasaccharide in an initial low-affinity complex. Two other residues, Lys 114 and Arg 129, then cooperate with Lys 125 to induce the low-affinity complex into an activated, high-affinity complex, in which a network of electrostatic interactions between antithrombin and the entire pentasaccharide is established. The identification of three critical basic residues in antithrombin and a trisaccharide in heparin as principal mediators of heparin activation of antithrombin may stimulate the design of small-molecule anticoagulants that mimic the action of heparin and are orally active.

Anticoagulants↗

PubMed2002

Heparin activates antithrombin anticoagulant function by generating new interaction sites (exosites) for blood clotting proteinases.

The plasma protein, antithrombin, and its polysaccharide activator, heparin, are essential anticoagulant regulators of blood clotting proteinases that are critical for maintaining hemostasis. Heparin activates antithrombin both by inducing conformational changes in the protein that specifically enhances factor Xa binding and by providing a surface to promote thrombin or factor Xa binding alongside antithrombin in a ternary bridging complex. Although x-ray structures of antithrombin, free and complexed with heparin, have suggested that exposure of a reactive proteinase binding loop is a key feature of conformational activation, mutagenesis of reactive loop residues indicates that the function of this structural change is not to present an optimal loop sequence to target clotting proteinases. Rather, the reactive loop sequence provides only the minimal requirements for recognition by either thrombin or factor Xa, and heparin activation enhances antithrombin recognition by these proteinases through the presentation of exosites outside of the reactive loop. These and other findings suggest that the reactive loop sequence of antithrombin was designed not for optimal recognition by procoagulant proteinases but rather to prevent recognition by the anticoagulant proteinase, activated protein C, thus ensuring that antithrombin functions as an effective anticoagulant.

Anticoagulants↗

PubMed1995

Inhibition of prothrombinase at macroscopic lipid membranes: competition between antithrombin and prothrombin.

The kinetics of inhibition of prothrombinase during prothrombin conversion by antithrombin and antithrombin-heparin complexes was studied in a tubular flow reactor. Prothrombinase was assembled at a macroscopic phospholipid membrane, composed of 25 mol % phosphatidylserine and 75 mol % phosphatidylcholine, deposited on the inner wall of a glass capillary, by perfusion with a factor Xa-factor Va mixture. Measurement of thrombin production allowed estimation of the amount of prothrombinase present at the capillary wall. Perfusion with a mixture of prothrombin and antithrombin or antithrombin-heparin complexes caused a progressive decline of the prothrombinase activity. The rate of inactivation steeply decreased with increasing prothrombin concentrations, indicating competitive inhibition. Analysis of competitive inhibition data requires estimation of the time-dependent substrate concentration, Co, near the prothrombin converting surface using earlier developed transport theory [Billy, D., et al. (1995) J. Biol. Chem. 270, 1029-1034]. It appears that the inhibition rate is proportional to the fraction of enzyme, Km/(Km+Co), not occupied by substrate. The value of Km of prothrombinase estimated from the dependence of the inhibition rate on the prothrombin concentration (Km = 2-3 nM) is in excellent agreement with the value estimated from the substrate conversion rate (Km = 3 nM). Therefore inhibition of prothrombinase by antithrombin and antithrombin-heparin complexes is fully competitive with the substrate: prothrombin. Our results show that prothrombinase assembled on macroscopic lipid surfaces by virtue of its low Km value is protected for inhibition due to highly effective competition of prothrombin with antithrombin for the active site of factor Xa.

Animals↗

PubMed1993

Immunologic evidence for insertion of the reactive-bond loop of antithrombin into the A beta-sheet of the inhibitor during trapping of target proteinases.

Identical or highly similar antigenic determinants, not present in the intact inhibitor, were induced in antithrombin on cleavage of the reactive bond, on formation of a complex between antithrombin and a synthetic reactive-loop tetradecapeptide, and on partial denaturation of antithrombin at low concentrations of guanidinium chloride. Previous studies indicate that the common structural feature of these three modified forms of antithrombin is that the region of the reactive-bond loop on the amino-terminal side of the reactive bond, or the corresponding synthetic peptide, is inserted as a middle strand in the main beta-sheet of the inhibitor, the A sheet. The new epitopes in the three modified antithrombin forms therefore most likely are exposed as a result of this insertion. Identical or highly similar epitopes were exposed also in complexes between antithrombin and thrombin or factor Xa, strongly suggesting that a substantial segment of the reactive-bond loop is inserted into the A sheet also in these complexes. In contrast, the new epitopes were not exposed in antithrombin on binding of heparin, implying that the conformational change induced by heparin does not involve such loop insertion. These results provide the first experimental verification of recent hypotheses that insertion of the reactive-bond loop of serpins into the A beta-sheet is involved in the binding of target proteinases.

Amino Acid Sequence↗

PubMed1986

Thrombin generation and inactivation in the presence of antithrombin III and heparin.

We have determined the rate constants of inactivation of factor Xa and thrombin by antithrombin III/heparin during the process of prothrombin activation. The second-order rate constant of inhibition of factor Xa alone by antithrombin III as determined by using the synthetic peptide substrate S-2337 was found to be 1.1 X 10(6) M-1 min-1. Factor Xa in prothrombin activation mixtures that contained prothrombin, and either saturating amounts of factor Va or phospholipid (20 mol % dioleoylphosphatidylserine/80 mol % dioleoylphosphatidylcholine, 10 microM), was inhibited by antithrombin III with a second-order rate constant that was essentially the same: 1.2 X 10(6) M-1 min-1. When both factor Va and phospholipid were present during prothrombin activation, factor Xa inhibition by antithrombin III was reduced about 10-fold, with a second-order rate constant of 1.3 X 10(5) M-1 min-1. Factor Xa in the prothrombin activation mixture that contained both factor Va and phospholipid was even more protected from inhibition by the antithrombin III-heparin complex. The first-order rate constants of these reactions at 200 nM antithrombin III and normalized to heparin at 1 microgram/mL were 0.33 and 9.5 min-1 in the presence and absence of factor Va and phospholipid, respectively. When the prothrombin concentration was varied widely around the Km for prothrombin, this had no effect on the first-order rate constants of inhibition. It is our conclusion that factor Xa when acting in prothrombinase on prothrombin is profoundly protected from inhibition by antithrombin III in the absence as well as in the presence of heparin.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1987

Inhibition of factor XIa by antithrombin III.

The inactivation of human factor XIa by human antithrombin III was studied under pseudo-first-order reaction conditions (excess antithrombin III) both in the absence and in the presence of heparin. The time course of inhibition was followed by using polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. After electrophoresis, proteins were blotted onto nitrocellulose and stained either for glycoprotein or for antithrombin III using antibodies against antithrombin III. Concomitant with factor XIa inactivation, two new slower migrating bands, one of which represented the intermediate complex consisting of one antithrombin III complexed with factor XIa, appeared as a transient band. Complete inactivation resulted in a single band representing the complex of factor XIa with two antithrombin III molecules. Quantitative analysis of the time course of inactivation was accomplished by measurement of the disappearance of factor XIa amidolytic activity toward the chromogenic substrate S2366. Pseudo-first-order reaction kinetics were observed throughout. The rate constant of inactivation was found to be 10(3) M-1 s-1 in the absence of heparin and 26.7 X 10(3) M-1 s-1 in the presence of saturating amounts of heparin. From the kinetic data, a binding constant (Kd) of 0.14 microM was inferred for the binding of antithrombin III to heparin. The time course of inactivation and the distribution of the reaction products observed upon gel electrophoresis are best explained assuming a mechanism of inactivation in which the two active sites present in factor XIa are inhibited in random order (i.e., independent of each other) with the same rate constant of inhibition.

Antithrombin III↗

PubMed2003

Crystal structure of antithrombin in a heparin-bound intermediate state.

Antithrombin is activated as an inhibitor of the coagulation proteases through its specific interaction with a heparin pentasaccharide. The binding of heparin induces a global conformational change in antithrombin which results in the freeing of its reactive center loop for interaction with target proteases and a 1000-fold increase in heparin affinity. The allosteric mechanism by which the properties of antithrombin are altered by its interactions with the specific pentasaccharide sequence of heparin is of great interest to the medical and protein biochemistry communities. Heparin binding has previously been characterized as a two-step, three-state mechanism where, after an initial weak interaction, antithrombin undergoes a conformational change to its high-affinity state. Although the native and heparin-activated states have been determined through protein crystallography, the number and magnitude of conformational changes render problematic the task of determining which account for the improved heparin affinity and how the heparin binding region is linked to the expulsion of the reactive center loop. Here we present the structure of an intermediate pentasaccharide-bound conformation of antithrombin which has undergone all of the conformational changes associated with activation except loop expulsion and helix D elongation. We conclude that the basis of the high-affinity state is not improved interaction with the pentasaccharide but a lowering of the global free energy due to conformational changes elsewhere in antithrombin. We suggest a mechanism in which the role of helix D elongation is to lock antithrombin in the five-stranded fully activated conformation.

Allosteric Site↗

PubMed1996

Antithrombin-heparin affinity reduced by fucosylation of carbohydrate at asparagine 155.

The two human plasma antithrombin isoforms, alpha and beta, differ in glycosylation at asparagine 135. Only the alpha form carries carbohydrate at this position and has lower affinity for heparin than the beta form. We previously found additional heterogeneity in a recombinant N135Q antithrombin variant, evidenced by two isoforms with a 2-fold difference in heparin affinity [Turko, I. V., Fan, B., & Gettins, P. G. W. (1993) FEBS Lett. 335, 9-12]. To test whether this heterogeneity of heparin affinity results from specific glycosylation differences, we have determined the carbohydrate composition at the three remaining glycosylation sites, asparagine residues 96, 155, and 192, in each of the two N135Q isoforms, by a combination of peptide fragmentation and electrospray mass spectrometry. Patterns of glycosylation at residues 96 and 192 were similar for each isoform and showed the presence of mono-, bi-, and triantennary complex carbohydrate, as well as fucosylation of all types of chains. At position 155, however, there was a marked difference between the isoforms, with the form with lower heparin affinity being 97% fucosylated at this position, whereas the form with higher affinity for heparin was not fucosylated. Other differences in carbohydrate type showed no strong correlation between the two isoforms. We conclude that formation of the two heparin-affinity isoforms of N135Q antithrombin results from the specific difference in fucosylation at residue 155, which may result in different structural properties of the carbohydrate. Consistent with these findings was the elimination of heparin-affinity heterogeneity in a double N135Q-N155Q variant antithrombin. It is possible that fucosylation of antithrombin may occur in vivo as a means of modifying the physiological properties of the antithrombin through alteration of the amount of antithrombin bound to surface heparin-like species.

Amino Acid Sequence↗

PubMed1999

The role of Arg46 and Arg47 of antithrombin in heparin binding.

Heparin greatly accelerates the reaction between antithrombin and its target proteinases, thrombin and factor Xa, by virtue of a specific pentasaccharide sequence of heparin binding to antithrombin. The binding occurs in two steps, an initial weak interaction inducing a conformational change of antithrombin that increases the affinity for heparin and activates the inhibitor. Arg46 and Arg47 of antithrombin have been implicated in heparin binding by studies of natural and recombinant variants and by the crystal structure of a pentasaccharide-antithrombin complex. We have mutated these two residues to Ala or His to determine their role in the heparin-binding mechanism. The dissociation constants for the binding of both full-length heparin and pentasaccharide to the R46A and R47H variants were increased 3-4-fold and 20-30-fold, respectively, at pH 7.4. Arg46 thus contributes only little to the binding, whereas Arg47 is of appreciable importance. The ionic strength dependence of the dissociation constant for pentasaccharide binding to the R47H variant showed that the decrease in affinity was due to the loss of both one charge interaction and nonionic interactions. Rapid-kinetics studies further revealed that the affinity loss was caused by both a somewhat lower forward rate constant and a greater reverse rate constant of the conformational change step, while the affinity of the initial binding step was unaffected. Arg47 is thus not involved in the initial weak binding of heparin to antithrombin but is important for the heparin-induced conformational change. These results are in agreement with a previously proposed model, in which an initial low-affinity binding of the nonreducing-end trisaccharide of the heparin pentasaccharide induces the antithrombin conformational change. This change positions Arg47 and other residues for optimal interaction with the reducing-end disaccharide, thereby locking the inhibitor in the activated state.

Amino Acid Substitution↗

PubMed2002

Designing small, nonsugar activators of antithrombin using hydropathic interaction analyses.

Conformational activation of antithrombin is a critical mechanism for the inhibition of factor Xa, a proteinase of the blood coagulation cascade, and is typically achieved with heparin, a polyanionic polysaccharide clinically used for anticoagulation. Although numerous efforts have been directed toward the design of better activators, a fundamental tenet of these studies has been the assumed requirement of an oligo- or a polysaccharide backbone. We demonstrate here a concept that small nonsaccharidic nonpolymeric molecules may be rationally designed to interact with and activate antithrombin for enhanced inhibition of factor Xa. The rational design strategy is based on a study of complexes of natural and mutant antithrombins with heparin-based oligosaccharides using hydropathic interaction (HINT) technique, a quantitative computerized tool for analysis of molecular interactions. A linear correlation was observed between the free energy of binding for antithrombinminus signoligosaccharide complexes and the HINT score over a wide range of approximately 13 kcal/mol, indicating strong predictive capability of the HINT technique. Using this approach, a small, nonsugar, aromatic molecule, (minus sign)-epicatechin sulfate (ECS), was designed to mimic the nonreducing end trisaccharide unit DEF of the sequence specific heparin pentasaccharide DEFGH. HINT suggested a comparable antithrombin-binding geometry and interaction profile for ECS and trisaccharide DEF. Biochemical studies indicated that ECS binds antithrombin with equilibrium dissociation constants of 10.5 and 66 microM at pH 6.0, I 0.025, and pH 7.4, I 0.035, respectively, that compare favorably with 2 and 80 microM observed for the natural activator DEF. ECS accelerates the antithrombin inhibition of factor Xa nearly 8-fold demonstrating for the first time that conformational activation of antithrombin is feasible with appropriately designed small nonsugar organic molecules. The results present unique opportunities for de novo activator design based on this first-generation lead.

Antithrombins↗

PubMed2001

Decreased plasma activity of antithrombin or protein C is not due to consumption coagulopathy in septic patients with disseminated intravascular coagulation.

We investigated whether depressed plasma antithrombin and protein C activity, considered as a specific finding of disseminated intravascular coagulation (DIC), is due to consumption coagulopathy in septic patients with DIC. An analysis of hemostatic parameters was performed in 139 septic patients (68 with DIC and 71 without DIC). Plasma activity of antithrombin and protein C tended to be significantly decreased in septic patients with DIC but not in those without DIC (p < 0.001). However, when the septic patients were classified into three groups according to the albumin (or choline esterase) level, no significant differences in antithrombin activity or protein C activity were observed between the patients with and without DIC in any of the subgroups. Notably, neither the plasma activity of antithrombin nor protein C was decreased even in septic patients with DIC who had normal plasma levels of albumin (or choline esterase). No significant correlation was observed between plasma levels of thrombin-antithrombin complex (TAT) and antithrombin activity, or between plasma levels of TAT and protein C activity either in septic patients with DIC or without DIC. It is reasonable to conclude that the markedly reduced plasma activity of antithrombin and protein C is not due to consumption coagulopathy in septic patients with DIC.

Aged↗

PubMed1981

Binding to antithrombin of heparin fractions with different molecular weights.

The interaction between bovine antithrombin, a plasma proteinase inhibitor, and heparin species of different molecular weights was studied. A commercial heparin preparation was divided by gel chromatography into a number of fractions with average molecular weights ranging from 6000 to 34700. Each of these fractions was further fractionated by affinity chromatography on matrix-bound antithrombin. In the latter procedure, those heparin fractions that had molecular weights lower than about 14000 were separated into three peaks. The material in the first of these was not adsorbed on the column, and the other two peaks corresponded to the low-affinity and high-affinity peaks described previously. In contrast, high-molecular-weight heparin samples gave only the low-affinity and high-affinity fractions. U.v. difference absorption studies showed that the non-adsorbed heparin fraction bound to antithrombin in solution with a binding constant at physiological ionic strength only slightly lower than that of low-affinity heparin. The division between the two fractions thus is arbitrary and only dependent on the conditions selected for the affinity-chromatography experiment. Stoicheiometries and binding constants for the binding of several high-affinity heparin species to antithrombin were determined by fluorescence titrations. High-affinity heparin fractions of equal elution positions in the beginning of the peaks of the affinity chromatographies, but with different molecular weights, showed stoicheiometries that were not experimentally distinguishable from 1:1 and also had no appreciable differences in binding constants. However, the anticoagulant activities, calculated on a molar basis, of these fractions increased markedly with molecular weight, a behaviour that thus cannot be explained by differences in the binding of the fractions to antithrombin. In contrast, high-affinity samples of similar molecular weights, which were eluted at increasing ionic strengths from matrix-linked antithrombin, were found to have an increasing proportion of chains with two binding sites for antithrombin and also to have progressively higher binding constants. These binding properties at least partly explain the increasing anticoagulant activities that were observed for these fractions.

Antithrombins↗

PubMed1981

Kinetic analysis of the heparin-enhanced plasmin--antithrombin III reaction. Apparent catalytic role of heparin.

Inactivation of plasmin by a 3-4-fold molar excess of antithrombin III follows pseudo-first-order kinetics and the apparent rate constants are proportional to the concentration of the inhibitor. Heparin accelerates the inactivation reaction without changing its pseudo-first-order character, and the apparent rate constants are also proportional to the concentration of the polysaccharide. Heparin results in a minimum 20-fold rate enhancement of the reaction between plasmin and antithrombin III when the concentrations of heparin and plasmin are approx. 0.5mum and 1mum respectively. Heparin at a molar concentration well below that of plasmin still accelerates the reaction: one molecule of the polysaccharide is able to facilitate the inactivation of about 100 molecules of plasmin. Heparin must bind to plasmin to accelerate the plasmin-antithrombin III reaction, since the modification of four to five lysine residues of the enzyme inhibits the rate-enhancement effect of heparin and the dissociation of heparin-plasmin complex decreases the inactivation rate of plasmin. Increasing the concentration of antithrombin III, at a constant amount of heparin, results in increase of the inactivation rate. By contrast, the effect of increasing the amount of plasmin in the presence of constant amount of heparin and antithrombin III is such that higher plasmin-to-heparin ratios are associated with lower rates of inactivation. It seems, therefore, that to obtain ;optimal' conditions for fast enzyme inactivation, the amount of heparin should be matched to plasmin rather than to antithrombin III. Arrhenius plots of the plasmin-antithrombin III reaction are linear both in the absence and presence of heparin, at concentrations of 1 or 2mug/ml, over a range of 26K. Under these experimental conditions, heparin increases activation entropy. The findings show that heparin seems to fulfil some criteria that are characteristic for biological catalysis: binding, reaction-rate enhancement (increasing activation entropy), recycling of heparin (effectiveness of non-stoichiometric amounts of the polysaccharide) and specificity.

Antithrombin III↗

PubMed1983

Properties of antithrombin-thrombin complex formed in the presence and in the absence of heparin.

Purification of antithrombin-thrombin complex by ion-exchange chromatography on DEAE-agarose resulted in predominantly monomeric complex, whereas purification on matrix-linked heparin produced large amounts of aggregated complex. Monomeric antithrombin-thrombin complexes formed in the presence and in the absence of heparin had similar conformations and heparin affinities. Moreover, the first-order dissociation rate constants, measured by thrombin release, of these complexes were similar, 2.3 X 10(-6)-3.4 X 10(-6)S-1, regardless of whether newly formed or purified complex was analysed. Similar dissociation rate constants were also obtained for purified complex formed with or without heparin, from analyses by dodecyl sulphate/polyacrylamide-gel electrophoresis of the release of modified antithrombin, cleaved at the reactive-site bond. No dissociation of intact antithrombin from the complex was detected by activity measurements or by gel electrophoresis. Aggregation of the complex was found to be accompanied by a decrease in apparent dissociation rate. The similar properties of antithrombin-thrombin complexes formed with or without heparin support the concept of a catalytic role for the polysaccharide in the antithrombin-thrombin reaction. Furthermore, the results indicate that the reaction between enzyme and inhibitor involves the rapid formation of an irreversible, kinetically stable, complex that dissociates into active thrombin and modified, inactive, antithrombin by a first-order process with a half-life of about 3 days. The inhibition thus resembles a normal proteolytic reaction, one intermediate step of which is very slow.

Animals↗

PubMed1992

Decreased affinity of recombinant antithrombin for heparin due to increased glycosylation.

Recombinant antithrombin produced by baby hamster kidney (BHK) or Chinese hamster ovary (CHO) cells was separated into two fractions, containing comparable amounts of protein, by affinity chromatography on matrix-linked heparin. Fluorescence titrations showed that the more tightly binding fraction had a heparin affinity similar to that of plasma antithrombin (Kd approximately 20 nM), whereas the affinity of the more weakly binding fraction was nearly 10-fold lower (Kd approximately 175 nM). Analyses of the heparin-catalysed rate of inhibition of thrombin further showed that the fractions differed only in their affinity for heparin and not in the intrinsic rate constant of either the uncatalysed or the heparin-catalysed inactivation of thrombin. The recombinant antithrombin fraction with lower heparin affinity migrated more slowly than both the fraction with higher affinity and plasma antithrombin in SDS/PAGE under reducing conditions, consistent with a slightly higher apparent relative molecular mass. This apparent size difference was abolished by the enzymic removal of the carbohydrate side chains from the proteins. Such removal also increased the heparin affinity of the weakly binding fraction, so that it eluted from matrix-linked heparin at a similar position to the deglycosylated tightly binding fraction or plasma antithrombin. Analyses of N-linked carbohydrate side chains showed that the weakly binding fraction from CHO cells had a higher proportion of tetra-antennary and a lower proportion of biantennary oligosaccharides than the tightly binding fraction. We conclude that the recombinant antithrombin produced by the two cell lines is heterogeneously glycosylated and that the increased carbohydrate content of a large proportion of the molecules results in a substantial decrease in the affinity of these molecules for heparin. These findings are of particular relevance for studies aimed at characterizing the heparin-binding site of recombinant antithrombin by site-directed mutagenesis.

Animals↗

PubMed1995

Elimination of glycosylation heterogeneity affecting heparin affinity of recombinant human antithrombin III by expression of a beta-like variant in baculovirus-infected insect cells.

In order to promote homogeneity of recombinant antithrombin III interactions with heparin, an asparagine-135 to alanine substitution mutant was expressed in baculovirus-infected insect cells. The N135A variant does not bear an N-linked oligosaccharide on residue 135 and is therefore similar to the beta isoform of plasma antithrombin. Purified bv.hat3.N135A is homogeneous with respect to molecular mass, charge and elution from immobilized heparin. Second-order rate constants for thrombin and factor Xa inhibition determined in the absence and presence of heparin are in good agreement with values established for plasma antithrombin and these enzymes. Based on far- and near-UV CD, bv.hat3.N135A has a high degree of conformational similarity to plasma antithrombin. Near-UV CD, absorption difference and fluorescence spectroscopy studies indicate that it also undergoes an identical or very similar conformational change upon heparin binding. The Kds of bv.hat3.N135A for high-affinity heparin and pentasaccharide were determined and are in good agreement with those of the plasma beta-antithrombin isoform. The demonstrated similarity of bv.hat3.N135A and plasma antithrombin interactions with target proteinases and heparins suggest that it will be a useful base molecule for investigating the structural basis of antithrombin III heparin cofactor activity.

Animals↗

PubMed2003

Probing plasma clearance of the thrombin-antithrombin complex with a monoclonal antibody against the putative serpin-enzyme complex receptor-binding site.

A high-affinity monoclonal antibody (M27), raised against the human thrombin-antithrombin complex, has been identified and characterized. The epitope recognized by M27 was located to the linear sequence FIREVP (residues 411-416), located in the C-terminal cleavage peptide of antithrombin. This region overlaps, by two residues, the putative binding site of antithrombin for the serpin-enzyme complex receptor. Studies in rats and with HepG2 cells in culture indicated that the Fab fragment of M27 does not block binding and uptake of the thrombin-antithrombin complex, suggesting that this region does not play a major role in the recognition and clearance of the thrombin-antithrombin complex. M27 blocked the ability of antithrombin to inhibit thrombin as well as antithrombin cleavage, both in the presence and absence of heparin.

Animals↗

PubMed1997

Functional requirements for inhibition of thrombin by antithrombin III in the presence and absence of heparin.

Mutation of 79 highly exposed amino acids that comprise approximately 62% of the solvent accessible surface of thrombin identified residues that modulate the inhibition of thrombin by antithrombin III, the principal physiological inhibitor of thrombin. Mutations that decreased the susceptibility of thrombin to inhibition by antithrombin III in the presence and absence of heparin (W50A, E229A, and R233A) also decreased hydrolysis of a small tripeptidyl substrate. These residues were clustered around the active site cleft of thrombin and were predicted to interact directly with the "substrate loop" of antithrombin III. Despite the large size of antithrombin III (58 kDa), no residues outside of the active cleft were identified that interact directly with antithrombin III. Mutations that decreased the susceptibility of thrombin to inhibition by antithrombin III in the presence but not in the absence of heparin (R89A/R93A/E94A, R98A, R245A, K248A, K252A/D255A/Q256A) in general did not also affect hydrolysis of the tripeptidyl substrate. These residues were clustered among a patch of basic residues on a surface of thrombin perpendicular to the face containing the active site cleft and were predicted to interact directly with heparin. Three mutations (E25A, R178A/R180A/D183A, and E202A) caused a slight enhancement of inhibition by antithrombin III.

Amino Acid Sequence↗